The present invention is generally in the field of methods for the detection of nucleic acid sequences through amplification and hybridization to labelled probes.
Hybridization methods are widely utilized in testing for the presence of particular nucleic acid sequences, identifying and/or quantifying such sequences. Hybridization occurs when there is sufficient complementarity of two nucleic acid sequences to effect non-covalent binding between the two molecules.
Various improvements and modifications have been introduced to increase the specificity and sensitivity of the reaction. In particular, polymerase chain reaction (PCR) techniques have been used to amplify the target nucleic acid sequence. In a recent development, two consecutive sets of PCR cycles ("nested PCR") have been used to both amplify and label the target nucleic acid sequence. The labelled sequence is then immobilized on a solid phase carrier, and testing is carried out using a reagent specific to the label. (See D J Kemp, et al., "Colorimetric detection of specific DNA segments amplified by polymerase chain reactions", Proc. Natl. Acad. Sci. USA 86, pp. 2423-2427, 1989).
WO89/11546 by Paulsen, et al., describes a method using PCR with DNA primers, one of which is soluble and the other which is bound to superparamagnetic particles, allowing greater ease of separation of the amplified product which facilitates detection. While the nucleic acid is bound to the particle, however, amplification via the beads is still taking place in a soluble phase, ie the beads are suspended in a liquid phase, increasing the time and complexity of the reaction, and requiring equipment for isolation of the superparamagnetic particles.
AU A-47144/89 by Molecular Diagnostics, Inc. also reports a method for increasing detection of nucleic acids using PCR to amplify sequences, where one or both of the primers are in solution or immobilized or immobilizable, for example, to Sephadex, to facilitate detection. The process can be used to yield immobilized target nucleic acid.
However, prior art methods for amplifying nucleic acid sequences have all suffered from problems associated with carrying out reactions in the liquid, rather than solid, phase. After each step of the reaction, the intermediate product must be isolated, before being reacted in the next step. Isolation steps are cumbersome, time consuming and often involve labile linkage reagents. In addition, no isolation step can be completely successful.
It is therefore an object of the present invention to provide a method which is rapid, simple, and provides an accurate, sensitive means for detecting small amounts of nucleic acid in a test sample.
It is a further object of the present invention to provide an assay which is relatively inexpensive, simple, rapid, and provides an accurate, sensitive means for detecting small amounts of nucleic acid in a test sample in clinical and research laboratories.